OP0077 SYNOVIAL RNA-SEQ ANALYSIS OF THE R4RA TRIAL IDENTIFIES SIGNATURES OF TREATMENT RESISTANCE AND REFRACTORY STATE IN RHEUMATOID ARTHRITIS
Authors: A. Surace, E. Sciacca, K. Goldmann, F. Rivellese, C. Cubuk, G. Giorli, L. Fossati-Jimack, M. Ahmed, E. Prediletto, M. Bombardieri, M. Lewis, C. Pitzalis
Journal: Annals of the Rheumatic Diseases (2022)

Background Although up to 5-20% of rheumatoid arthritis (RA) patients do not respond to all current medications including biologic therapies, relatively little is known about the underlying pathogenic mechanisms driving non-response. In the first biopsy-driven randomized clinical trial in RA (R4RA) 1 , patients, in whom synthetic-DMARDs and at least one anti-TNF drug were not effective, were randomised 1:1 to rituximab (RTX) or tocilizumab (TOC) with a balanced stratification based on their synovial B-cell rich/poor signature, and response was assessed at 16 weeks. Non-responders were subsequently allowed to switch to the alternative drug with 48-week follow-up. Objectives Investigate mechanisms of response and non-response to RTX and TOC through deep molecular (RNA-Sequencing) profiling of synovial tissue. Methods RNA-Seq from baseline synovial tissue biopsies of patients who received RTX (n=88) or TOC (n=94) at any point in the trial was analysed for differentially expressed genes and associated modules between responders and non-responders. Response was defined as 50% improvement in clinical disease activity index (CDAI) score. Patients who had received both drugs during the trial were subdivided into RTX only responders (pro-RTX, n=9), TOC only responders (pro-TOC, n=12) and refractory patients (no response to both RTX &amp; TOC, n=32) and analysed for differential gene expression and performed gene module analysis. Results 6625 genes were significantly differentially expressed between RTX responders compared to non-responders, with a predominance of antigen presentation as well as T- and B-cell genes being associated with response, while non-response was linked to fibroblast associated genes. Comparison between TOC responders and non-responders identified fewer (85) differentially expressed genes, however lymphocyte and immunoglobulin genes were also high in the synovial tissue of TOC responders similar to RTX responders, while non-responder genes and modules also included a fibroblast signature. The cross-over study design enabled comparison of rituximab-specific responders (pro-RTX), tocilizumab-specific responders (pro-TOC) and refractory patients (non-responders to both RTX &amp; TOC, n=32) in a 3-way analysis (see Figure 1). This identified 1980 genes upregulated both in pro-RTX and pro-TOC patients, 175 genes exclusive to the pro-RTX group and 306 to the pro-TOC group, while 1277 genes were exclusive to the refractory group. While leukocyte modules and genes dominated RTX &amp; TOC response, the refractory state was strongly associated with fibroblast genes and modules. We confirmed the observed expansion of fibroblasts from the RNA-Seq data by immunohistochemistry showing the presence of DKK3+ sublining fibroblasts in refractory rather than responder patients. Conclusion We provide novel insights into the cellular and molecular pathways underpinning multi-biologic resistance that define a refractory RA phenotype, characterised by a stromal/fibroblast signature. References [1]Humby, F. , et al. Lancet (2021) Acknowledgements We would like to thank all patients and the R4RA recruiting centres and principal investigators http://www.r4ra-nihr.whri.qmul.ac.uk/recruiting_centres.php We would also like to acknowledge the UK National Institute of Health Research for funding the R4RA trial (grant reference: 11/100/76) and Versus Arthritis for providing infrastructure support through the Experimental Arthritis Treatment Centre (grant number: 20022). Disclosure of Interests None declared.